The Actinobacteria or Actinomycetes are a phylum of Gram-positive bacteria. Most are found in the soil, playing an important role in decomposition of organic materials such as cellulose and chitin. Other Actinobacteria colonize plants and animals, and include a few pathogens such as Mycobacterium. Representative genera include: Actinomyces, Arthrobacter, Bifidobacterium, Corynebacterium, Frankia, Micrococcus, Micromonospora, Mycobacterium, Nocardia, Propionibacterium, and Streptomyces. 
The actinobacteria are unsurpassed in their ability to produce compounds that have pharmaceutical activity. As early as 1940, Selman Waksman discovered that the soil bacteria he was studying made the antibiotic “actinomycin” and was awarded the Nobel Prize for his work. Since then hundreds of naturally occurring antibiotics have been discovered in these terrestrial microorganisms, especially from the genus Streptomyces. Therefore, this phylum is a very important source of medicinal compounds, and there is always a need to maximize the production of such pharmaceuticals.
One way of improving the production of any compound made by bacteria is through metabolic engineering—the purposeful re-design of an organism's metabolic pathways by recombinant DNA techniques. Metabolic engineering has the potential to considerably improve process productivity and is increasingly used in both academic and industrial institutions. Most current metabolic engineering studies have focused on manipulating enzyme levels. However, cofactors also play an essential role in a large number of biochemical reactions and their manipulation has the potential to be used as an additional tool to achieve desired metabolic engineering goals.
For example, Coenzyme A (CoA) is an essential cofactor in numerous reactions and is involved in the regulation of key metabolic enzymes. In fact, it has been estimated that as much as 4% of all enzymes utilize CoA, CoA thioesters, or 4′-phosphopantetheine as substrates. Further, CoA compounds, including acetyl-CoA, propionyl-CoA, malonyl-CoA, and methylmalonyl-CoA, are essential precursors required for the biosynthesis of polyketides and other complex biomolecules.
Polyketides are small, cyclized molecules. Between 5000 and 10,000 are known, and about 1% of them possess drug activity. Sales of the more than 40 polyketide drugs—including antibiotics, immunosuppressants, cholesterol-lowering agents, antifungals, and cancer chemotherapeutics—exceed $15 billion a year. But polyketides are difficult to synthesize chemically, and the exotic microbes that produce them naturally can be hard to grow in culture.
Sugars are the structural components of different types of natural products. Important antibiotics, antifungals, antiparasites and anticancer drugs possess sugars attached to the aglycon core. These sugar components participate in the molecular recognition of the cellular target by the bioactive compound and, therefore, its presence is important, in many cases essential, for the biological activity of many natural products. A great majority of these sugars belong to the 6-deoxyhexoses (6DOHs). These sugars are synthesized from nucleoside diphosphate-activated hexoses (mainly D-glucose) via a 4-keto-6-deoxy intermediate. Two common enzymatic steps leading to the biosynthesis of this intermediate are catalyzed by a dNDP-D-hexose synthase and dNDPD-hexose-4,6-dehydratase. The different 6DOHs will vary depending on the substituents and/or the stereochemistry at carbon atoms at positions 2, 3, 4, or 5 of the hexose carbon chain, resulting from deoxygenations, transaminations and/or C, N, or O methylations. D- and L-isomeric forms of many 6DOHs exist as a result of the action of a 5- or a 3,5-epimerase.
In recent years, a number of 6DOHs gene clusters have increasingly been characterized, most of them participating in the biosynthesis of different antibiotics produced by Actinomycetes. Deoxy sugar carrying glycosides and polysaccharides are believed to be secondary gene products, which are synthesized from activated sugars by glycosyltransferases. The activation is brought about in analogy to mammalian pathways by nucleoside diphosphates. It is the activated sugar that is derivatized in general before being transferred to the respective aglycon. The deoxysugars are transferred to the corresponding aglycon by glycosyltransferases, which are generally sugar-, aglycon-, and site-specific. In recent years, increasing evidence has suggested some degree of “flexibility” of glycosyltransferases involved in the biosynthesis of secondary metabolites, and there have been some reports of examples in which different deoxysugars have been transferred by a glycosyltransferase to its aglycon. One of these glycosyltransferases, the elloramycin glycosyltransferase (ElmGT), has been shown to be especially “flexible” in accepting different L- and D-deoxysugars and also being able to transfer a disaccharide. Recently, several plasmids that direct the biosynthesis of L-daunosamine, L-Olivose, L-oleandrose or D-desosamine have been reported by the different groups. These plasmids contain different subsets of genes involved in the biosynthesis of these deoxysugars from several antibiotic-producing organisms.
What is needed in the art are metabolic engineering methods that can be applied to Actinomyces, such as Streptomyces, to increase the production of polyketides and other complex biomolecules.
E. coli have been already been metabolically engineered to increase CoA production (4), whereby a 10 fold increase in CoA and a 5 fold increase in acetyl-CoA was seen on overexpression of the panK gene and supplementation with pantothenic acid. However, the metabolic engineering of E. coli is not easily applied to the Actinomycetes, which is a much more difficult bacterial phylum to engineer (2). Further, early attempts were only made to improve the production of primary CoA products and acetyl-coA, and improved production of secondary metabolites was not attempted.